CN102972297B - Method for cultivating regeneration plants of cotton - Google Patents
Method for cultivating regeneration plants of cotton Download PDFInfo
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- CN102972297B CN102972297B CN201210518521.7A CN201210518521A CN102972297B CN 102972297 B CN102972297 B CN 102972297B CN 201210518521 A CN201210518521 A CN 201210518521A CN 102972297 B CN102972297 B CN 102972297B
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Abstract
The invention discloses a method for cultivating regeneration plants of cotton. The method for cultivating target cotton plants, provided by the invention, comprises the following steps of: (1) firstly, removing stem tips, branches and leaves as well as fibrous roots of a target cotton plant, and retaining 1-2 white fibrous roots or basal parts of the fibrous roots to obtain a main body; and secondly, cutting off along a first section, which is close to the root part, of the main body and retaining the first section, provided with the root, of the main body to obtain a plant to be subjected to subculture; (2) culturing the plant to be subjected to subculture in a regeneration plant subculture medium to obtain a regeneration plant subjected to subculture again; and (3) hardening and transplanting the regeneration plant subjected to the subculture again to obtain the transplanted cotton plant, wherein the target cotton plant is the regeneration cotton plant obtained after tissue culture. Proven by experiments, the cultivation method for cultivating the regeneration plants of cotton has the capability of greatly increasing the survival rate of transplanting of the regeneration plants of cotton. The method disclosed by the invention is simple to operate, strong in practicability and is conductive to improving the genetic transformation efficiency of the cotton.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the cultivation method of a kind of cotton regenerated plant.
Background technology
Cotton is the important source material of textile industry, fine chemistry industry, is also important strategic materials.Transgene cotton is one of crop of realizing the earliest commercialization plantation.2011, transgene cotton reached 2,470 ten thousand hectares in global cultivated area, accounted for the more than 80% of 3,000 ten thousand hectares of the cotton gross areas.
Utilize biotechnology that merit gene is proceeded in cotton, can break species boundary, expand genetic resources.
At present, by biotechnology, cultivate the conventional genetic transforming method of transgene cotton new varieties and have agrobacterium-mediated transformation, micropellet bombardment method and pollen tube passage method etc.
Agriculture bacillus mediated method for transformation is by means of natural Agrobacterium, plants wound to be infected to the ability that produces crown gall nodule or send out shape root to improve a kind of biology techniques forming.The genetic transformation of cotton mostly adopts the Agrobacterium tumefaciems (containing Ti-plasmids) after improvement, but different strains is also different to the affinity of different cotton varieties, directly affects transformation efficiency.The applicable explant of agriculture bacillus mediated genetic transformation is very extensive, comprises blade, stem section, plumular axis, petiole, cotyledon, rataria, callus or mature seed, but the most frequently used on cotton be hypocotyl.
Micropellet bombardment method is again via Particle Bombardment Transformation method, utilizes power by directly injecting in recipient cell after the particulate acceleration of parcel DNA, foreign DNA can be contacted with cytogenetics material, and then be incorporated in genome.During micropellet bombardment, conventional acceptor is shoot apical meristem, embryo callus and suspended culture cell.
Pollen tube passage method is early 1980s period-luminosity space proposition the earliest.Initial is mainly, by injection, the recombination gene building or plasmid are injected to ovary.Be further developed into afterwards column cap semar technique and pollen and carried method.
These Cotton Transformation methods have all obtained success.But what be wherein most widely used is still agriculture bacillus mediated retransmission method, has obtained the transformation event of applying in a plurality of production.It has following characteristics: transformation mechanism is comparatively clear, and the exogenous dna fragment of conversion is 50kb, and foreign gene is incorporated on chromosome with single copy or low copy form, and genetic stability is good.But application the method need to first be set up complete tissue and cultivate and plant regeneration system, also only in the part kind of jade-like stone word cotton, sets up at present, and length consuming time, and transformation efficiency is lower.
The transplanting efficiency that improves the resistant plant of regeneration will contribute to improve the genetic transformation efficiency of cotton.Be mainly by the method for acclimatization and transplants after directly taking root at present, or transplant by the method for grafting.The method and technology of grafting is strong, higher to operator's requirement.And transplant by hardening after directly taking root, easily cause a large amount of resistance transplantation of seedlings unsuccessful, because in organizing for a long time cultivation, resistance screening and atomization, many regeneration plants become a little less than growing ability, root system is undeveloped, even vitrifying.Therefore a kind of set up simple and feasible raising transplanting efficiency method is very necessary.
Summary of the invention
The object of this invention is to provide a kind of method of cultivating object cotton plants.
Method provided by the invention, comprises the steps:
1) first remove stem apex, branches and leaves and the fibrous root of object cotton plants, retain the fibrous root of 1-2 root white or retain fibrous root base portion, obtain trunk; Along described trunk, first joint place near root disconnects again, retains the trunk first segment with root, obtains the plant for the treatment of that subculture is cultivated;
2) by described, treat that plant that subculture cultivates is at regeneration plant subculture medium relaying culture, regeneration plant after obtaining again subculture and cultivating;
3) described subculture is again cultivated to rear regeneration plant through hardening, transplanting, obtained transplanting rear cotton plants;
The regeneration cotton plants of described object cotton plants for obtaining after tissue is cultivated.
In said method, step 2) in, described subculture is cultivated as subculture cultivation 1-2 time.
In said method, step 2) in, described regeneration plant subculture medium is to contain the MSB solid culture medium that final concentration is 0.1mg/l NAA.
MSB solid culture based formulas is as shown in embodiment.
In said method, step 2) in, described condition of culture is that 33 ℃ of light of temperature are cultivated 16 hours, 25 ℃ of dark cultivations 8 hours of temperature; The intensity of illumination that above-mentioned light is cultivated is specially 2000LUX.
In said method, described transplanting comprises the steps:
1) will move into and be equipped with in the container of soil through the plant of hardening, then bagging or cover preservative film moisturizing one week;
2) will be through 1) plant of processing removes bag or preservative film, cultivates, and obtains transplanting rear cotton plants.
In said method, described soil is that seedling medium (moisten female board, fully stocked wood flowers market is bought) mixes with 1/2 volume vermiculite.
In said method, the described regeneration cotton plants obtaining after tissue is cultivated is the regeneration cotton plants obtaining after the regeneration cotton plants obtaining after cultivating by agriculture bacillus mediated tissue or the tissue mediating by micropellet bombardment method are cultivated.
The regeneration cotton plants that above-mentioned method of cultivating by agriculture bacillus mediated tissue obtains carries out according to one of embodiment 1;
The above-mentioned tissue mediating by micropellet bombardment method is cultivated the regeneration cotton plants obtaining and is carried out according to one of embodiment 2.
Of the present invention experiment showed, adopts method of the present invention to transplant cotton regeneration plant, can greatly improve the transplanting survival rate of cotton regenerated plant.The present invention is simple to operate, practical, contributes to improve the genetic transformation efficiency of cotton.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Component in following embodiment in MSB medium is as shown in table 1, and liquid MSB medium is all water-soluble for the final concentration shown in the solute of table 1 is pressed, and adds glucose to 30 grams/l of final concentration preparation and obtains.Solid MSB medium is to add curing agent phytagel to final concentration 2.5g/l in liquid MSB medium.
Table 1 is the component in MSB medium
| Macroelement | Final concentration (gL in medium -1） |
| NH 4NO 3 | 1.65 |
| KNO 3 | 1.9 |
| KH 2PO 4 | 0.17 |
| MgSO 4.7H 2O | 0.37 |
| CaCl 2 | 0.44 |
| Trace element | Concentration (mgL in medium -1） |
| FeSO 4.7H 2O | 27.8 |
| Na 2EDTA | 37.3 |
| MnSO 4.4H 2O | 22.3 |
| ZnSO 4.4H 2O | 8.6 |
| H 3BO 3 | 6.2 |
| KI | 0.83 |
| Na 2MoO 4.2H 2O | 0.25 |
| CuSO 4.5H 2O | 0.025 |
| CoCl 2.6H 2O | 0.025 |
| Organic principle | Concentration (mgL in medium -1） |
| Glycine | 2.0 |
| Thiamine hydrochloride | 10 |
| Pyridoxine hydrochloride | 1 |
| Nicotinic acid | 1 |
| Inositol | 100 |
The transplanting of embodiment 1, agriculture bacillus mediated acquisition regeneration cotton plants
One, agriculture bacillus mediated Cotton Transformation obtains regeneration cotton plants
In the case that report succeeds at present, the acceptor kind of cotton is mainly that jade-like stone word is cotton, but also comprises some other kind, as CCRI 12, cotton No. 3 of nasal mucus, cotton No. 7 of Shanxi, anti-No. 6 of China etc., India cultivation cotton variety Anjli (LRK516) and LRA 5166 etc.Genetic transformation mostly adopts the Agrobacterium tumefaciems (containing Ti-plasmids) after improvement, as LBA4404, and PGV2260, EMH105 etc., but different strains is also different to the affinity of different cotton varieties, directly affects transformation efficiency.The explant using is very extensive, comprises blade, stem section, plumular axis, petiole, cotyledon, rataria, callus or mature seed, but the most frequently used is still hypocotyl.The medium component using in conversion process, hormone types, condition of culture etc. are all not quite similar.
The cotton acceptor kind research that new explant is set up High-Efficient Cotton conversion system for Coker312(utilizes that embodiments of the invention are concrete used; Cotton Science, 2002,14(1): 22～27.; Public Ke Cong Biological Technology institute, Chinese Academy of Agricultural Sciences obtains), Agrobacterium be LBA4404(purchased from sky, Beijing bounties company, article No. is 12-96), cotton acceptor is hypocotyl.Concrete steps are as follows:
1) Aseptic seedling culture: pour a small amount of concentrated sulfuric acid lint in Coker312 seed into, the alcohol sterilizing 5min with 75%, then soaks 30 minutes sterile water wash 3 times with the liquor natrii hypochloritis of 1.8% valid density.The good seed of sterilizing is soaked in sterile water, and 37 ℃ are spent the night.The seed of opening is peelled off to kind of a skin, plant in 1/2MSB medium, secretly cultivate 9 days, 28 ℃ of temperature, obtain aseptic seedling.
2) preparation of Agrobacterium bacterium liquid
To contain object plasmid pCambia3301(purchased from Cambia company) Agrobacterium LBA4404 bacterium liquid be inoculated into 50ml containing in the YEB liquid nutrient medium of 100mg/L kanamycin, 28 ℃, 220rpm shaking table is cultured to OD 0.6 left and right.Bacterium liquid, in the centrifugal 5min of 5,000rpm, is abandoned to supernatant, and precipitation is resuspended with the MSB liquid nutrient medium that has added 100mg/L acetosyringone, obtains Agrobacterium bacterium liquid.
3) infect and be total to cultivation
Remove 1) root and the cotyledon of the aseptic seedling that obtains, the segment that hypocotyl is cut into 1cm left and right is explant, by 2) the Agrobacterium bacterium liquid that obtains adds in explant, places 20min, during shake several times, the explant after being infected;
Explant after taking-up is infected, blots with the filter paper of sterilizing, is then placed in common medium, and 15 explant/wares, secretly cultivate 72h, and 22 ℃ of temperature, obtain the explant after common cultivation.
Above-mentioned altogether medium is prepared as follows: adding final concentration is 0.1mg/l KT, 0.1mg/l 2,4-D and 50mg/l acetosyringone MSB solid culture medium.
4) callus of induce and subculture are cultivated
(1) induction is cultivated
By 3) explant after common cultivation that obtains proceeds in inducing culture, and condition of culture is illumination 16 hours, and dark 8 hours, 28 ℃ of temperature, can see callus and produce after 1 month, obtain inducing rear callus;
Above-mentioned inducing culture is prepared as follows: add 0.1mg/l KT, 0.1mg/l 2, the MSB solid culture medium of 4-D, 500mg/Lcef, 25mg/L kan.
(2) after the induction above-mentioned (1) being obtained, callus subculture every about 1 month is cultivated 1 time, and subculture is 5 times altogether, obtains embryo callus; The condition that subculture is cultivated is consistent with above-mentioned induction condition of culture;
1st, 2 subculture mediums are prepared as follows: add 0.1mg/l KT, 0.1mg/l 2, the MSB solid culture medium of 4-D, 500mg/Lcef, 50mg/L kan
The 3rd time later subculture medium is prepared as follows: add 0.01mg/l KT, 0.01mg/l 2, the MSB solid culture medium of 4-D, 100mg/L kan;
5) differentiation is cultivated and embryoid induction
By above-mentioned 4) embryo callus that obtains proceeds in differential medium, and condition of culture is illumination 16 hours, dark 8 hours, 28 ℃ of temperature; Cultivate 2 months, until embryoid forms.
Differential medium is prepared as follows: the MSB solid culture medium of the KT that to add final concentration be 0.15mg/l, the IBA that final concentration is 0.5mg/l, kan that final concentration is 50mg/L.
6) cotyledonary embryos with become bud induction to cultivate
By 5) the embryoid callus that obtains proceeds to bud inducing culture, and condition of culture is illumination 16 hours, and dark 8 hours, 28 ℃ of temperature, cultivated 3 months, until budlet output.
Bud inducing culture is prepared as follows: the MSB solid culture medium of the KT that to have added final concentration be 0.15mg/l, the IBA that final concentration is 0.5mg/l, Gln (glutamine) that final concentration is 1.0mg/l, Asn (asparagine) that final concentration is 0.5mg/l, kan that final concentration is 100mg/L.
7) seedling root media
By above-mentioned 6) obtain grow to the budlet that 4cm is high, proceed to take in the MSB solid that the sucrose of 30mg/l is carbon source and take root, condition of culture is illumination 16 hours, dark 8 hours, 28 ℃ of temperature, cultivated 3 months, obtain the cotton plants of regenerating.
50 seeds, through above-mentioned cultivation, are obtained to 42 strain regeneration cotton plants.
Two, regeneration cotton plantlet of transplant
All adopt following method to process above-mentioned 42 strain regeneration cotton plants:
1, regeneration plant subculture is cultivated
1) process
21 strain regeneration cotton plants are all first cut to stem apex and branches and leaves, remove the fibrous root of whole brown, retain the fibrous root of 1-2 root white or the base portion of reservation fibrous root, obtain trunk; Along trunk, near first joint place of root, cut off trunk again, retain the trunk section with root, obtain the plant for the treatment of that subculture is cultivated;
2) subculture is cultivated
By 1) in treating of the obtaining plant access regeneration plant subculture medium that subculture cultivates, by 33 ℃ of light, cultivate (intensity of illumination is 2000LUX) 16 hours, 25 ℃ of dark cultivations of temperature 8 hours; Cultivate the plant that obtains again taking root about 1 month; This subculture plant can grow to 5cm height left and right, trunk increasing amount thick, root system increases and color is white or yellow-white.
Regeneration plant subculture medium is to contain the MSB solid culture medium that final concentration is 0.1mg/l NAA.
By the subculture cultivation (condition of culture and medium are constant) again of the above-mentioned plant again taking root, obtain again subculture and cultivate rear regeneration plant.
2, practice seedling and transplanting
1) practice seedling
To contain above-mentioned 1 obtain grow to about 5cm height again subculture after cultivating, pour the sterile water that 2cm is thick in the blake bottle of regeneration plant, sealed membrane is unclamped but do not remove completely, in incubator, place 3 days, by illumination (intensity of illumination is 2000LUX), cultivate 16 hours 33 ℃ of temperature, dark 8 hours, 25 ℃ of cultivations of temperature, then remove sealed membrane completely, then place 3 days, during this time can be suitable add some sterile waters, obtain practicing plant after seedling.
2) transplant
The soil using during transplanting is seedling medium (moisten female board, fully stocked wood flowers market is bought), wherein adds the vermiculite of 1/2 volume.Transplant and evening before that day soil is irrigated, the humidity that is placed into soil the next morning is just suitable.
After practicing seedling, plant is taken out gently together with medium, with clear water, by medium wash clean, puts into flowerpot soil, mulching soil is also real soil pressure, on flowerpot, with suction pipe, cost support, preservative film or foil-type plastic bag moisturizing in covering, and on plastic sack, prick hole to breathe freely; Flowerpot is moved in incubator and placed for 1 week, and condition of culture is that light is cultivated 16 hours (intensity of illumination is 2000LUX), 33 ℃ of temperature, dark 8 hours, 25 ℃ of cultivations of temperature.Then remove preservative film or foil-type plastic bag, continue to place one week.Within in above-mentioned transplanting process 4 ~ 5 days, water 30ml water, with soil dry being as the criterion not too.Again flowerpot is moved on in greenhouse and cultivated, cultivate 30 days, statistics survival strain number, obtains cotton plants after the well-grown transplanting of 21 strain altogether.
Contrast: 21 strains regeneration cotton plants are cultivated without above-mentioned two 1 regeneration plant subculture, directly practiced seedling, transplanting, cotton plants after obtaining 12 strains and transplanting.
Can find out, the plant survival rate after subculture is cultivated has improved a lot.
Embodiment 2, the mediation of micropellet bombardment method obtain the transplanting of regeneration cotton plants
One, the mediation of micropellet bombardment method obtains regeneration cotton plants
Particle bombardment while carrying out Cotton Transformation conventional acceptor have shoot apical meristem, embryo callus and suspended culture cell.The cotton acceptor kind that report succeeds has upland cotton and sea-island cotton.The internal factor of recipient cell and particle gun bombardment parameters can affect the transformation efficiency of foreign gene.The factor of acceptor self comprises before and after the potential regeneration capacity of physiological status, the cell of explant kind, cell, bombardment the processing of cell and intracellular environment the ability to accept of foreign DNA etc.Particle gun bombardment parameters generally will be considered dispersive range, degree and the bombardment number of times of bombarding pressure, target distance, micro-bullet.Before bombardment and after bombardment, cell is carried out to suitable infiltration processing can increase the chance that foreign DNA enters cell, the slight plasmolysis that this processing simultaneously causes can reduce kytoplasm seepage, improves transformant survival rate.
The cotton acceptor kind that embodiments of the invention are concrete used is Coker312, and acceptor material is the embryo callus that hypocotyl produces; Concrete steps are as follows:
1) Aseptic seedling culture: identical with one 1 the method for embodiment 1;
2) callus of induce and subculture are cultivated:
Remove 1) root and the cotyledon of the aseptic seedling that obtains, the segment that hypocotyl is cut into 1cm left and right is explant;
Explant is proceeded in inducing culture, and condition of culture is illumination 16 hours, and dark 8 hours, 28 ℃ of temperature, can see callus and produce after 1 month, obtain inducing rear callus;
Above-mentioned inducing culture is prepared as follows: adding final concentration is 0.1mg/l KT, 0.1mg/l 2, the MSB solid culture medium of 4-D.
The subculture every about 1 month of callus after induction obtained above is cultivated 1 time, and subculture is 5 times altogether, obtains embryo callus; The condition that subculture is cultivated is consistent with above-mentioned induction condition of culture with medium component;
3) micropellet bombardment
(1) embryo callus is inoculated into height and oozes on solid culture medium and process 4 hours, 28 ℃ of temperature, obtain callus to be bombarded;
Height oozes solid culture medium and is prepared as follows: the MSB solid culture medium that adds final concentration 0.2mol/L mannitol+0.2mol/L sorbierite.
(2) pCambia3301 plasmid being concentrated into concentration is 1 μ g/ μ l;
(3) bronze is processed
1. 50mg 1.0 μ m bronzes are placed in to 1.5ml import centrifuge tube;
2. add 1ml sterile water, abundant vortex, the centrifugal 10s of 10000r/m, removes supernatant, sterile water repeated washing 3 times;
3. use the resuspended bronze grain of aseptic 50% glycerine of 1ml, the bronze solution after processing can be preserved at-20 ℃ long-term;
(4) micro-bullet preparation
The bronze solution that adds 30 μ l to handle well in aseptic import 1.5ml EP pipe, the plasmid DNA of 10 μ lpCambia3301,100 μ l 2.5mol/L CaCl
2, 40 μ l 0.1mol/L spermidines, vortex concussion 1min, is placed in rapidly on ice, so repeats 10 times.Be placed on ice more than 30min.In the centrifugal 15s of 12000r/m, remove supernatant.Add 400 μ l 70% ethanol, mix, in the centrifugal 15s of 12000r/m, remove supernatant.Resuspended with 80 μ l absolute ethyl alcohols (HPLC), obtain micro-bullet absolute ethyl alcohol suspension.
(5) micropellet bombardment
Used is Bio-Rod PDS-1000/He type particle gun;
1. get the micro-bullet absolute ethyl alcohol of 15 μ l suspension, put in carrier film center, dry;
2. open particle gun and helium valves, helium pressure is set.Installation can be split film, carrier film;
3. it is appropriate location that the culture dish that callus to be bombarded is housed (1) being obtained is put in, and making target distance is 9cm, in 1100psi, bombards callus twice, obtains bombarding rear sample.
4) be total to cultivation, screening and culturing
By 3) (5) bombardment of obtaining after sample at height, ooze on solid culture medium and continue to process 16h, then proceed on inducing culture and cultivate 7 days, then proceed to screening and culturing base and cultivate 2 months, obtain callus after subculture; Above-mentioned condition of culture is all illumination 16 hours, dark 8 hours, and 28 ℃ of temperature.
Screening and culturing base is prepared as follows: adding final concentration is 0.1mg/l KT, 0.1mg/l 2, the MSB solid culture medium of 4-D, 50mg/Lkan;
5) differentiation is cultivated and embryoid induction
With above-described embodiment 1 one 5) method identical;
6) cotyledonary embryos with become bud induction to cultivate
With above-described embodiment 1 one 6) method identical;
7) seedling root media
With above-described embodiment 1 one 7) method identical, obtain the cotton plants of regenerating.
50 seeds, through above-mentioned cultivation, are obtained to 139 strain regeneration cotton plants.
Two, regeneration cotton plantlet of transplant
All adopt following method to process above-mentioned 69 strain regeneration cotton plants:
1, the subculture of regeneration plant is cultivated
1) process: identical with the method for embodiment 1;
2) subculture is cultivated: identical with the method for embodiment 1.
2, practice seedling and transplanting
1) practice seedling: identical with the method for embodiment 1;
2) transplant: identical with the method for embodiment 1;
Obtain 68 strains and transplant rear cotton.
Contrast: the subculture of 1 the regeneration plant by 69 strains regeneration cotton plants without above-mentioned two is cultivated, and directly practices seedling, transplanting, cotton after obtaining 42 strains and transplanting.
Can find out, the plant survival rate after subculture is cultivated improves a lot.
Claims (2)
1. a method of cultivating object cotton plants, comprises the steps:
1) first remove stem apex, branches and leaves and the fibrous root of object cotton plants, retain the fibrous root of 1-2 root white or retain fibrous root base portion, obtain trunk; Along described trunk, first joint place near root disconnects again, retains the trunk first segment with root, obtains the plant for the treatment of that subculture is cultivated;
2) by described, treat that plant that subculture cultivates is at regeneration plant subculture medium relaying culture, regeneration plant after obtaining again subculture and cultivating; Described regeneration plant subculture medium is to contain the MSB solid culture medium that final concentration is 0.1mg/l NAA; Described condition of culture is that 33 ℃ of light of temperature are cultivated 16 hours, 25 ℃ of dark cultivations 8 hours of temperature; Described subculture is cultivated as subculture cultivation 2 times; The intensity of illumination that above-mentioned light is cultivated is 2000LUX;
3) described subculture is again cultivated to rear regeneration plant through hardening, transplanting, obtained transplanting rear cotton plants;
The regeneration cotton plants of described object cotton plants for obtaining after tissue is cultivated;
Above-mentioned MSB solid culture medium is: the final concentration shown in the component in MSB medium is pressed is all water-soluble, and adds glucose and prepare and obtain the component in MSB medium to 30 grams/l of final concentration; Solid MSB medium is to add curing agent phytagel to final concentration 2.5g/l in liquid MSB medium;
Component in MSB medium is as described below:
Macroelement:
Trace element:
Organic principle:
2. method according to claim 1, is characterized in that:
Described transplanting comprises the steps:
1) will move into and be equipped with in the container of soil through the plant of hardening, then bagging or cover preservative film moisturizing one week;
2) will be through 1) plant of processing removes bag or preservative film, cultivates, and obtains transplanting rear cotton plants.
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| CN104956930A (en) * | 2015-07-14 | 2015-10-07 | 山西省农业科学院棉花研究所 | Method for suspended transplantation of regenerated transgenic cotton plant |
| CN106801065B (en) * | 2015-11-25 | 2021-01-26 | 华中农业大学 | Method for improving cotton regeneration and transformation efficiency and application |
| CN107950340A (en) * | 2017-11-01 | 2018-04-24 | 河北省农林科学院遗传生理研究所 | A kind of cotton test tube method for transplanting of high-survival rate |
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| GB9915071D0 (en) * | 1999-06-28 | 1999-08-25 | Zeneca Ltd | Regeneration of cotton plants |
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| CN1312981C (en) * | 2005-04-30 | 2007-05-02 | 山西省农业科学院棉花研究所 | Substrate and method of cotton regeneration test tube seedling direct transplantation |
| CN1907001A (en) * | 2005-08-03 | 2007-02-07 | 中国农业科学院棉花研究所 | Cuttage reproducing method for cotton |
| CN100429305C (en) * | 2005-08-03 | 2008-10-29 | 中国农业科学院棉花研究所 | Rhizogenesis culture method of cotton regenerating seeding and special culture medium thereof |
| CN100496225C (en) * | 2006-06-27 | 2009-06-10 | 中国农业科学院棉花研究所 | Cotton leafstalk tissue cultivation and high-differentiation rate material selective breeding method |
| CN102057869B (en) * | 2009-11-18 | 2012-09-26 | 中国农业科学院棉花研究所 | Cotton regeneration seedling rooting method and special culture medium thereof |
| CN101773039B (en) * | 2010-01-28 | 2011-05-25 | 山西省农业科学院棉花研究所 | Transgenic cotton regenerated plant transplantation method |
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